Allergology International xxx (2018) 1e7

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Allergology International
journal homepage: http://www.elsevier.com/locate/alit

Original Article

Effects of anti-allergic drugs on T cell-mediated nasal

hyperresponsiveness in a murine model of allergic rhinitis
Tomoe Nishimura a, *, Osamu Kaminuma a, b, c, Mayumi Saeki a, Noriko Kitamura a,
Minoru Gotoh a, Akio Mori b, Takachika Hiroi a
a
Allergy and Immunology Project, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan
b
Clinical Research Center for Allergy and Rheumatology, National Hospital Organization, Sagamihara National Hospital, Kanagawa, Japan
c
Center for Life Science Research, University of Yamanashi, Yamanashi, Japan

a r t i c l e i n f o a b s t r a c t

Article history: Background: We have recently demonstrated that T cell-mediated nasal hyperresponsiveness (NHR) is a
Received 28 January 2018 representative pathophysiological feature of allergic rhinitis (AR). Although several anti-allergic drugs
Received in revised form are used for the treatment of AR, the efficacy of these drugs on T cell-mediated NHR have not been
14 April 2018
elucidated. In these studies we investigated the effects of dexamethasone (Dex), montelukast (Mk), and
Accepted 1 May 2018
Available online xxx
chlorpheniramine (Chl) on NHR in antigen-immunized and antigen-specific Th2 cell-transferred mice.
Methods: OVA-immunized BALB/c mice were treated with Dex, Mk, or Chl and challenged intranasally
with OVA. We then assessed NHR, the number of inflammatory cells in the nasal lavage fluid (NALF),
Keywords:
Anti-allergic drugs
mRNA expression of Th2 cytokines in the nasal tissue, the population of CD3þCD4þ cells in the nasal
Allergic rhinitis lymphoid tissue (NALT), and antigen-specific serum IgE and IgG levels. Antigen-induced NHR and changes
CD4þ T cell in antigen-specific T cells in the NALT were investigated in OVA-specific Th2 cell-transferred mice.
Dexamethasone Results: Dex significantly suppressed antigen-induced NHR, inflammatory cell infiltration, and IL-4, IL-5,
Th2 IL-6, and IL-13 expression in immunized mice. Chl was completely ineffective, and only IL-13 expression
was suppressed by Mk. None of these drugs affected IgE and IgG production. Antigen-induced NHR
Abbreviations: and the increase in antigen-specific T cells in the NALT of Th2 cell-transferred mice were inhibited by
AR, allergic rhinitis; BHR, bronchial Dex, but not by Mk or Chl.
hyperresponsiveness; BSA, bovine serum Conclusions: Steroids are effective for the reduction of NHR in AR by suppressing the accumulation of
albumin; Chl, chlorpheniramine; Cys- inflammatory cells, especially antigen-specific T cells.
LT, cysteinyl leukotriene;
Copyright © 2018, Japanese Society of Allergology. Production and hosting by Elsevier B.V. This is an open access
Dex, dexamethasone; Mk, montelukast;
article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
NAL, nasal lavage; NALF, nasal lavage fluid;
NALT, nasal lymphoid tissue; NHR, nasal
hyperresponsiveness; TCR, T cell receptor

Introduction symptoms in response to non-specific stimuli is accompanied by

Allergic rhinitis (AR) is characterized by several nasal symptoms
including sneezing, rhinorrhea, and nasal congestion following
provocation by a specific antigen. The submucosal accumulation of
inflammatory cells, including eosinophils, neutrophils, and T cells
are also found in the nasal tissues of AR patients.1,2 Furthermore,
nasal hyperresponsiveness (NHR) represented by increased nasal
* Corresponding author. Allergy and Immunology Project, Tokyo Metropolitan
Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo 156-8506,
Japan.
E-mail address: nishimura-tm@igakuken.or.jp (T. Nishimura).
Peer review under responsibility of Japanese Society of Allergology.
these inflammatory responses.3,4
Steroids and receptor antagonists for leukotrienes and hista-
mines are used as anti-allergic drugs for the treatment of AR.5,6
Histamine H1 receptor antagonists reduce histamine-induced
nasal symptoms such as sneezing and rhinorrhea, but they are
considered less effective for nasal congestion. In contrast, cysteinyl
leukotriene (Cys-LT) 1 receptor antagonists have been reported to
relieve nasal congestion. Steroids are used for severe AR patients,
because they exhibit a strong anti-inflammatory effect. However, the
effects of these drugs against NHR have not been investigated. This
is due, in part, to the lack of an animal model with the pathophysi-
ological features of NHR in AR.
We have recently demonstrated that the antigen-specific CD4þ
T cells play a crucial role in the development of NHR using murine
models of AR.7 Antigen-induced NHR in immunized mice was

https://doi.org/10.1016/j.alit.2018.05.002
1323-8930/Copyright © 2018, Japanese Society of Allergology. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/
licenses/by-nc-nd/4.0/).

Please cite this article in press as: Nishimura T, et al., Effects of anti-allergic drugs on T cell-mediated nasal hyperresponsiveness in a murine
model of allergic rhinitis, Allergology International (2018), https://doi.org/10.1016/j.alit.2018.05.002
2 T. Nishimura et al. / Allergology International xxx (2018) 1e7

reduced by depleting CD4þ T cells using an anti-CD4 antibody. By employing these models, this study evaluated the effects
Furthermore, normal mice transferred with in vitro-differentiated of dexamethasone (Dex, a steroid), montelukast (Mk, a Cys-LT1
antigen-specific T cells developed significant NHR following receptor antagonist), and chlorpheniramine (Chl, a histamine H1
antigen challenge. receptor antagonist) in a murine model of antigen-induced NHR.

Fig. 1. Effects of anti-allergic drugs on antigen-induced allergic responses in OVA-immunized mice. Antigen-immunized mice were treated with Dex, Mk, or Chl 30 min before OVA
challenge. (A) Six hours after the last challenge, NHR was evaluated by counting the number of sneezes evoked by BSA administration. (B) Subsequently, the number of inflam-
matory cells in the NALF and (C) the expression of IL-4, IL-5, IL-6, and IL-13 mRNA in the nasal tissue was measured. (D) Antigen-specific serum IgE and IgG were measured by ELISA.
Data are expressed as means ± SEM of 4e6 mice. * p < 0.05, ** p < 0.01, and *** p < 0.001 as compared with OVA-challenged control mice (Dunnett's method).
Chl, chlorpheniramine; Dex, dexamethasone; Mk, montelukast; NALF, nasal lavage fluid; NHR, nasal hyperresponsiveness.

Please cite this article in press as: Nishimura T, et al., Effects of anti-allergic drugs on T cell-mediated nasal hyperresponsiveness in a murine
model of allergic rhinitis, Allergology International (2018), https://doi.org/10.1016/j.alit.2018.05.002
 T. Nishimura et al. / Allergology International xxx (2018) 1e7 3

We investigated the mechanisms of these drugs by examining
inflammatory cells in the nasal lavage fluid (NALF), cytokine
expression in the nasal tissue, serum IgE and IgG levels, and
antigen-specific CD4þ T cells in the nasal lymphoid tissue (NALT).
Methods
Animals
Six to eight week old female BALB/c mice were obtained from
Japan SLC (Shizuoka, Japan). DO11.10/RAG-2/ mice, which ex-
press the OVA-specific T cell receptor (TCR) ab, were maintained for
antigen-specific Th2 cell preparation as previously described.8 The
experimental protocols were approved by the Animal Use and Care
Committee of the Tokyo Metropolitan Institute of Medical Science.
Anti-allergic drugs
Dex, Mk sodium hydrate (TCI, Tokyo, Japan), and Chl maleate
salt (SigmaeAldrich, MO, USA) were dissolved in ethanol or PBS,
diluted with PBS to a final ethanol concentration of 10.0e33.3%,
and then injected subcutaneously at 10, 30, and 100 mg/kg,
respectively. The vehicle alone did not affect any parameters
examined in this study.
In vitro polarization of antigen-specific TH2 cells
Antigen-specific Th2 cells were prepared as previously
described.8,9 Briefly, OVA-specific naïve CD4þ T cells were isolated
from splenocytes of DO11.10/RAG-2/ mice by positive selection
using CD4 microbeads and magnetic cell sorting (Miltenyi, Bergisch
Gladbach, Germany). Cells were cultured with X-ray-irradiated
splenocytes in Dulbecco's Modified Eagle-nutrient mixture
F12-HAM medium (SigmaeAldrich) supplemented with 10% fetal
bovine serum. At the start of culture, 0.3 mM synthetic OVA323-339
peptide, 10 U/ml recombinant IL-2 (Shionogi, Osaka, Japan), 10 U/ml
recombinant IL-4 (PeproTech, NJ, USA), and 10 mg/ml antieIFNeg
monoclonal antibody (R4-6A2, eBioscience, CA, USA) were added.
Seven days after stimulation, cells were harvested and used for the
adoptive transfer.
Antigen-induced NHR
In the antigen-immunization model, BALB/c mice were sensi-
tized by an intraperitoneal injection of 20 mg OVA (Hyglos GmbH,
Regensburg, Germany) emulsified with 2.25 mg alum adjuvant
(Thermo Fisher Scientific, MA, USA) on days 0, 7, 14, and 21 of the
experiment. Every day on days 35e39, mice were challenged with
an intranasal administration of 600 mg OVA dissolved in 20 ml PBS or
PBS alone. For pharmacological studies, Dex, Mk, and Chl were
administrated subcutaneously 30 min before antigen challenge on
days 35e38. In the Th2 cell transfer model, polarized Th2 cells
(2  107) were intravenously injected into each BALB/c mouse on
day 0, and these mice were intranasally challenged with OVA or
PBS daily on days 1e4. Dex, Mk, and Chl were administrated
subcutaneously 30 min before antigen challenge on days 1e3.
NHR was assessed 6 h after the last challenge. Histamine is
frequently used as a stimulant for evaluating NHR, though it was not
suitable to evaluate the effect of Chl on NHR. We already confirmed
that several proteins including bovine serum albumin (BSA) and
casein can be used as non-specific stimulants for evaluating
NHR.7 Therefore, we assessed NHR by counting the number of

Fig. 2. Effects of anti-allergic drugs on the antigen-induced increase in CD4þ T cells in the NALT. Antigen-immunized mice were treated with Dex, Mk, or Chl 30 min before OVA
challenge. (A) Six hours after the last challenge, the CD3þCD4þ T cell population in the NALT was determined by flow cytometry. (B) Percentage of CD3þCD4þ T cells in NALT. Data
are expressed as means ± SEM of 4e6 mice are shown. ** p < 0.01 as compared with OVA-challenged control mice (Dunnett's method). Chl, chlorpheniramine; Dex, dexamethasone;
Mk, montelukast; NALT, nasal lymphoid tissue.

Please cite this article in press as: Nishimura T, et al., Effects of anti-allergic drugs on T cell-mediated nasal hyperresponsiveness in a murine
model of allergic rhinitis, Allergology International (2018), https://doi.org/10.1016/j.alit.2018.05.002
4 T. Nishimura et al. / Allergology International xxx (2018) 1e7

Please cite this article in press as: Nishimura T, et al., Effects of anti-allergic drugs on T cell-mediated nasal hyperresponsiveness in a murine
model of allergic rhinitis, Allergology International (2018), https://doi.org/10.1016/j.alit.2018.05.002
 T. Nishimura et al. / Allergology International xxx (2018) 1e7 5

sneezes following intranasal administration of 30 mg/ml BSA for
5 min (SigmaeAldrich).
In some experiments, sneezing responses were also assessed
immediately after the 3rd OVA challenge in immunized mice
and following administration of OVA instead of BSA in Th2
cell-transferred mice. These models did not show any inflammatory
features in the lower airways.10
Evaluation of antigen-induced nasal inflammation
Following the assessment of NHR, mice were euthanized by
administrating a lethal dose of sodium pentobarbital (Sumitomo
Dainippon Pharma, Osaka, Japan). Subsequently, inflammatory
cells in the nasal lavage fluid (NALF) were collected from antigen-
immunized mice and classified by morphology as previously
described.11 At the same time, peripheral blood was recovered and
antigen-specific serum IgE and IgG levels were measured by
ELISA using horseradish peroxidase-conjugated anti-mouse IgE
(Serotech, Oxford, UK) and IgG (Southern Biotech Associates, Bir-
mingham, AL, USA) antibodies, respectively.12 Serum dilutions used
in the ELISA were 1:100 and 1:1000 for IgE and IgG, respectively.
For several mice, total RNA was extracted from the nasal tissue
around the nasal concha approximately 6 mm from the top of
the nose in a 3  3 mm square and 2 mm thick. After reverse tran-
scription using random primers (Toyobo, Osaka, Japan) and Super-
Script III reverse transcriptase (Thermo Fisher Scientific), quantitative
real-time RT-PCR for IL-4, IL-5, IL-6, and IL-13 was performed using
Assay-on-DemandTM Gene Expression Products (TaqMan® MGB
probes) with an CFX96™ Real-Time PCR Detection System (Bio-Rad,
Hercules, CA, USA) as previously described.13
NALT cell population assay
NALT cells were prepared as previously described.14 Briefly,
the lower jaw and the tongue were removed from euthanized
mice, and the NALT was carefully excised by peeling the upper
palate from the incisors teeth side toward the molar teeth side. The
collected NALT was mechanically mashed followed by filtration
through 35 mm filter. The NALT cell population was evaluated by
flow cytometry using anti-CD4-APC eFluor780 (Thermo Fisher
Scientific), anti-CD3-PECy7, and anti-DO11.10-TCR-PE (BioLegend,
CA, USA) monoclonal antibodies. Rat IgG2a (k)-APCeFluor780
(Thermo Fisher Scientific), rat IgG2b (k)-PECy7, and mouse IgG2a
(k)-PE (BioLegend) were used as isotype-matched negative control
antibodies, respectively.
Statistical analysis
The results are presented as the means ± SEM. Statistical
analysis was performed using a one-way ANOVA and Dunnett's
multiple comparison test. p < 0.05 was statistically significant.
Results
Effects of anti-allergic drugs on antigen-induced NHR and nasal
inflammation in immunized mice
OVA-immunized mice were treated with Dex, Mk, and Chl
and then challenged with OVA. Following the repeated antigen
challenge, the development of NHR was observed as a significant
increase in BSA-induced sneezing response as compared to the
PBS-challenged control mice (Fig. 1A). NHR was significantly sup-
pressed by the administration of Dex, but not Mk or Chl (Fig. 1A),
whereas antigen-induced sneezing response observed immediately
after the 3rd OVA challenge was suppressed by Chl and Dex
(Supplementary Fig. 1). Antigen-induced increases in NALF
lymphocytes, neutrophils, and eosinophils were significantly sup-
pressed only by Dex (Fig. 1B).
The expression of IL-4, IL-5, IL-6, and IL-13 mRNA in nasal tissue
was augmented by antigen challenge (Fig. 1C). Dex significantly
downregulated these cytokine genes, and the same trend was
observed in Mk-treated mice, but IL-13 expression was the only
cytokine that was significantly suppressed. No significant effects on
cytokine expression were observed in Chl-treated mice.
Although serum OVA-specific IgE and IgG levels were signifi-
cantly elevated in antigen-immunized and -challenged mice, they
were not affected by any of the anti-allergic drugs tested (Fig. 1D).
Under this experimental condition, OVA-specific IgE and IgG were
not detectable in naïve mice (data not shown).
Since we have proven that CD4þ T cells play a central role in
inducing NHR in our experimental AR model,7 the effects of anti-
allergic drugs on CD4þ T cells in the NALT were examined by flow
cytometry for CD3þCD4þ cells in antigen-immunized and -chal-
lenged mice. A significantly larger population of CD4þ T cells was
seen in the OVA-challenged mice as compared to PBS-challenged
control mice (Fig. 2A, B). The administration of Dex, but not Mk
or Chl slightly reduced the CD4þ T cell population.
Effects of anti-allergic drugs on antigen-induced NHR and antigen-
specific T cell accumlation in TH2 cell-transferred mice
To elucidate the effects of anti-allergic drugs on antigen-specific
T cells, we used a recently established Th2 cell-transferred mouse
model.7 OVA-specific Th2 cells were created from DO11.10/RAG-2/

splenocytes by in vitro stimulation cell culture. In BALB/c mice
adoptively transferred with Th2 cells, significant NHR developed
following OVA challenge (Fig. 3A). Similar to the results in the
antigen-immunization model, the NHR was significantly sup-
pressed by the administration of Dex, but not Mk or Chl (Fig. 3A). In
contrast, antigen-induced sneezing response was significantly
suppressed by Chl (Supplementary Fig. 2).
Since there was a trend toward a decrease in NALT CD4þ T cells
induced by Dex treatment in antigen-immunized mice (Fig. 2),
antigen-specific T cells in the NALT were investigated by moni-
toring the CD4þDO11.10-TCRþ population in Th2 cell-transferred
mice. Significantly more antigen-specific T cells were seen in
OVA-challenged mice as compared to PBS-challenged control mice
(Fig. 3B, C). The administration of Dex, but not Mk or Chl, signifi-
cantly reduced the change in the antigen-specific T cell population.
Discussion
AR is characterized by several symptoms such as sneezing,
rhinorrhea, and nasal congestion. A variety of anti-allergic drugs
including steroids and receptor antagonists for leukotrienes and
histamines are used to suppress these symptoms. However, the
efficacy of these drugs on NHR associated with AR symptoms has
not been elucidated. In this study, antigen-induced NHR

Fig. 3. Effects of anti-allergic drugs on antigen-specific Th2 cell-induced nasal responses. OVA-specific Th2 cell-transferred mice were treated with Dex, Mk, or Chl 30 min before
OVA challenge. (A) Six hours after the last challenge, NHR was evaluated by counting the number of sneezes evoked following BSA administration. (B) The CD4þDO11.10-TCRþ
population in the NALT was assessed by flow cytometry. (C) Percentage of CD4þDO11.10-TCRþ cells in NALT. Data are expressed as means ± SEM of 4e6 mice are shown. * p < 0.05
and ** p < 0.01 as compared with OVA-challenged control mice (Dunnett's method). Chl, chlorpheniramine; Dex, dexamethasone; Mk, montelukast; NALT, nasal lymphoid tissue;
NHR, nasal hyperresponsiveness.

Please cite this article in press as: Nishimura T, et al., Effects of anti-allergic drugs on T cell-mediated nasal hyperresponsiveness in a murine
model of allergic rhinitis, Allergology International (2018), https://doi.org/10.1016/j.alit.2018.05.002
6 T. Nishimura et al. / Allergology International xxx (2018) 1e7
accompanied by nasal Th2 cytokine expression, an influx of antigen-
specific T cells were suppressed by Dex.
The correlation between the effects of anti-allergic drugs on
antigen-induced NHR and the decrease in antigen-specific T cells is
consistent with our previous investigation demonstrating the
crucial contribution of CD4þ T cells to NHR.7 Taken together, these
studies demonstrated that NHR was diminished by the depletion of
CD4þ T cells in antigen-immunized mice and that significant NHR
developed in antigen-specific T cell-transferred mice.7 Typically, AR
patients experience an increase in CD4þ T cells in their nasal
mucosa, and this can be downregulated by steroid treatment.15,16
The reduction in antigen-specific T cells may be an essential
mechanism in the steroid-mediated suppression of NHR.
Steroids display strong inhibitory effects on a variety of T cell
activities including T cell growth, differentiation, cytokine produc-
tion, and chemotaxis.17 Their suppression of T cell proliferative and
chemotactic activity may indicate the mechanism by which Dex
reduces antigen-specific T cells in the NALT. Furthermore, reports
show that the CCR4 preferentially expressed on Th2 cells plays an
important role in the pathogenesis of AR.18,19 The expression of CCR4
on peripheral lymphocytes in patients with bronchial asthma is
reduced following steroid treatment.20 Therefore, intrinsic chemo-
tactic activity and chemokine receptor expression may be involved
in the migration of antigen-specific T cells during steroid treatment.
Although our results suggest that histamine has a negligible role in
the development of NHR, the IgE-mast cell axis has the potential to
accumulate antigen-specific Th2 cells by releasing cyclooxygenase
metabolites.21 Steroid treatment has been reported to reduce the
number of mast cells in the mucosal tissues.22 Further studies are
required to clarify the relative contributions of these mechanisms to
the suppression of T cell-mediated NHR by Dex.
Steroids also exhibited other inhibitory effects on T cell activity.
Consistent with previous clinical and animal studies,23,24 antigen-
induced cytokine production in the nasal tissue was inhibited by
Dex in antigen-immunized mice. IL-5 and IL-6 play a crucial role in
the local migration of eosinophils and neutrophils.25,26 Moreover, the
chemotactic activity of eosinophils and neutrophils are suppressed
by steroids in vitro.27,28 This suggests that the significant suppression
of eosinophil and neutrophil infiltration in the nasal mucosa by Dex
was caused by the downregulation of the chemotactic activity of
these inflammatory cells and cytokine production from T cells.
Interestingly, only IL-13 expression was suppressed by Mk.
IL-13 is a multifunctional cytokine released by T cells and other in-
flammatory cells.29 IL-13 is essential for IgE synthesis, goblet cell
hyperplasia, mucus hypersecretion, fibrosis, and chitinase upregu-
lation.29 Although the contribution of IL-13 to the development of
bronchial hyperresponsiveness (BHR) has been reported in animal
models of asthma,30e32 our findings clearly suggest that IL-13 as well
as IgE/IgG are not essential for T cell-mediated NHR. The reason for
this discrepancy is unclear, although the contribution of IL-13 to the
development of BHR and NHR is possibly different. With respect to
the role of IL-13 in BHR, this cytokine promotes the proliferation of
airway smooth muscle cells.33 However, NHR is characterized by
a certain increment of sneezes and that this is induced by neural
reflexes that are unaffected changes to smooth muscle cells. Since we
have demonstrated that Th1 and Th17 cells did not produce IL-13 but
induced NHR, a mechanism different from that of IL-13-mediated
BHR may participate in the development of NHR.
Although the statistical significance was not seen, Mk tended to
suppress antigen-induced nasal IL-5 expression. This result is partly
supported by a previous report indicating that the suppression of
IL-5 by Mk in a rat asthma model.34 We have demonstrated the
importance of IL-5-mediated eosinophilic inflammation in Th2-
mediated BHR.35e38 However, we recently found that eosinophils
are dispensable for NHR developed in antigen-immunized mice.7
Please cite this article in press as: Nishimura T, et al., Effects of anti-allergic drugs on T cell-mediated nasal hyperresponsiveness in a murine
model of allergic rhinitis, Allergology International (2018), https://doi.org/10.1016/j.alit.2018.05.002
Given that NHR is inducible by Th1 and Th17 cells, IL-5 may
not play a major role in the development of NHR, at least under our
experimental conditions.
Several allergic symptoms can be induced in an IgE-
independent manner and the effectiveness of anti-histamines for
such symptoms has been reported.39,40 Interestingly, antigen-
induced sneezing response in Th2 cell-transferred mice was
significantly suppressed by Chl, even though this agent was not
effective for the NHR. Iwasaki et al. demonstrated that the sneezing
response evoked by antigen plus lipopolysaccharide in Th2
cell-transferred mice was mediated by histamine derived from
monocytes and/or macrophages.40 The contribution of those cells
to antigen-induced NHR deserves further examination.
In summary, steroids have suppressive effects on T cell-mediated
NHR by reducing antigen-specific T cells. The intra- and intercellular
signaling cascades involved in the development of NHR may be the
next targets in the generation of new anti-allergic drugs.
Acknowledgements
This work was supported by research grants from the ‘Agri-
Health Translational Research Project’ from the 1) Ministry of
Agriculture, Forestry and Fisheries of Japan to Hiroi T, 2) a Grant-in-
Aid for Scientific Research from the Japan Society for the Promotion
of Science (KAKENHI, Grant Number 15K07787) to Kaminuma O, 3)
the Tojuro Iijima Foundation for Food Science and Technology to
Kaminuma O, 4) Nipponham Foundation for the Future of Food to
Kaminuma O, and 5) Mishima Kaiun Memorial Foundation to
Kaminuma O.
Appendix A. Supplementary data
Supplementary data related to this article can be found at
https://doi.org/10.1016/j.alit.2018.05.002.
Conflict of interest
The authors have no conflict of interest to declare.
Authors' contributions
Conceived and designed the experiments: TN and OK. Performed the experi-
ments: TN, MS, and NK. Analyzed the data: TN and OK. Contributed reagents, mice,
materials, and analytical tools: MG, AM and TH. Wrote the paper: TN and OK. All
authors read and approved the final manuscript.
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